The fusion of peptides to the coat proteins of amplifiable genetic particles, e.g., phage, is a widely used method for screening combinatorial libraries of peptides (Rodi and Malowski, Curr. Opin. Biotechno., 10:87–93 (1999); Wilson and Finlay, Canadian Journal of Microbiology, 44:313–329 (1998)). One common approach is to express random sequences at the N-terminus of the bacteriophage M13 coat protein pIII, resulting in library complexities of up to 109 different clones. Selection is achieved by performing multiple rounds of target binding (panning), elution and amplification. Each round of panning enriches the pool of clones in favor of the tightest-binding ligands. Because each phage particle contains both the displayed peptide and the DNA encoding it, the selected peptides can be readily identified by DNA sequencing. Despite its utility and convenience, in vivo biological expression limits library diversity to combinations of twenty of the naturally occurring amino acids, linked by peptide bonds.
This problem can be partially circumvented by taking advantage of the enormous potential chemical diversity of synthetic combinatorial libraries. A vast body of work has been carried out with libraries consisting of systematic variations of peptides (Geysen, et al., Proc. Natl. Acad. Sci. USA, 81:3998–4002 (1984); Houghten, et al., Nature 354:84–86 (1991), Lam, et al., Nature, 354: 82–84 (1991)), peptide analogues (Figliozzi, et al., Methods Enzymol., 267:437–447 (1996), and small molecules (Bunin, et al., Methods Enzymol., 267:448–465 (1996), and an entire industry has been built around this type of combinatorial chemistry. While libraries well in excess of 1018 different molecules (equivalent to 1 μmol of material if one molecule of each variant is present) can be synthesized, the identification of which molecules bind to a given target from such a vast pool is problematic. Libraries are typically synthesized in spatially addressable form, e.g., grids of pins or wells each containing one compound (Geysen, supra), or tethered to macromolecular beads containing a chemical tag which specifically identifies the attached compound (Lam, et al., supra). Ligand identification thus limits the size of chemically synthesized libraries to a practical upper limit of 104–106 different molecules. Unlike biosynthetic libraries such as phage display peptide libraries, however, chemically synthesized libraries are not limited to a small subset of potential functional diversity.
The functional diversity of phage displayed peptide libraries can be increased by specifically chemically modifying the library prior to each round of panning. Phage libraries with enzymatically phosphorylated tyrosine residues have been constructed to map protein kinase and SH2 domain recognition sequences (Dente, et al., Journal of Molecular Biology, 269:694–703 (1997); Schmitz, et al., J. Mol. Biol., 260:664–677 (1996)). Phage libraries have also been biotinylated at specific lysine residues during in vivo phage morphogenesis, but this method requires a specific 66-residue biotinylation motif (Stolz, et al., FEBS Lett., 440:213–217 (1998)). Both of these methods require defined flanking sequence, and the incorporated modification cannot be altered. Therefore neither are generally applicable to incorporation of any desired chemical functionality in the context of a randomized amino acid sequence. For example, there are no methods for specifically modifying displayed tyrosine with other chemical moieties while protecting endogenous tyrosine residues elsewhere on the phage coat. The side chains of lysine and cysteine are reactive, but small-molecule reagents are likely to target residues within the native coat protein in addition to the displayed peptide. A new type of phage library, with a unique site available for a broad range of chemical modifications, is therefore needed.
To maintain the essential amplification and selection techniques of phage display, the existing bacterial genetic machinery should be employed to incorporate the unique reactive site into the displayed peptide. A method in which a non-native residue is incorporated into a phage-displayed protein by native chemical ligation (Dwyer, et al., Chem. Biol., 7:263–274 (2000)) could in principle be used to incorporate a unique reactive site, but this method requires that the non-native residue be incorporated within a synthetic peptide sequence, which is then chemically ligated onto a phage displayed polypeptide. As a result, the residues flanking the potential modification site are not encoded on the phage genome, severing the link between displayed sequence and DNA sequence.